ab75743 was affinity purified from rabbit antiserum by affinity chromatography using epitope specific phosphopeptide. The antibody against non phosphopeptide was removed by chromatography using non phosphopeptide corresponding to the phosphorylation site.

Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

WB

1/500 - 1/1000. Predicted molecular weight: 59 kDa.

IHC-P

1/50 - 1/100.

Target

Function

Tyrosine protein phosphatase which functions as a dosage-dependent inducer of mitotic progression. Directly dephosphorylates CDK1 and stimulates its kinase activity. Also dephosphorylates CDK2 in complex with cyclin E, in vitro.

Sequence similarities

Belongs to the MPI phosphatase family.Contains 1 rhodanese domain.

Domain

The phosphodegron motif mediates interaction with specific F-box proteins when phosphorylated. Putative phosphorylation sites at Ser-79 and Ser-82 appear to be essential for this interaction.

Post-translationalmodifications

Phosphorylated by CHEK1 on Ser-76, Ser-124, Ser-178, Ser-279, Ser-293 and Thr-507 during checkpoint mediated cell cycle arrest. Also phosphorylated by CHEK2 on Ser-124, Ser-279, and Ser-293 during checkpoint mediated cell cycle arrest. Phosphorylation on Ser-178 and Thr-507 creates binding sites for YWHAE/14-3-3 epsilon which inhibits CDC25A. Phosphorylation on Ser-76, Ser-124, Ser-178, Ser-279 and Ser-293 may also promote ubiquitin-dependent proteolysis of CDC25A by the SCF complex. Phosphorylation of CDC25A at Ser-76 by CHEK1 primes it for subsequent phosphorylation at Ser-79, Ser-82 and Ser-88 by NEK11. Phosphorylation by NEK11 is required for BTRC-mediated polyubiquitination and degradation. Phosphorylation by PIM1 leads to an increase in phosphatase activity. Phosphorylated by PLK3 following DNA damage, leading to promote its ubiquitination and degradation.Ubiquitinated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase complex that contains FZR1/CDH1 during G1 phase leading to its degradation by the proteasome. Ubiquitinated by a SCF complex containing BTRC and FBXW11 during S phase leading to its degradation by the proteasome. Deubiquitination by USP17L2/DUB3 leads to its stabilization.

Thank you for your reply. I am sorry that the antibodies are still not producing good results in WB. We do not currently have new lots available for either product, so I have issued you a refund (Credit Note: ***). To redeem this refund, please have your purchasing agent contact our accounting department at us.credits@abcam.com. Please let me know if you have any further questions.

Thank you for contacting us. I am sorry that these antibodies are giving weak signal in your samples.

For Cyclin B2, I would expect ab18250 to give good staining using the protocol you sent, however this protein is not very highly expressed in normal or cancerous liver. You may find better results testing a positive control such as bone marrow or HeLa cells.

The antibody ab75742 is phospho-specific, so I would not recommend using milk as a blocking agent. Milk contains endogenous phosphatases which can cleave the phosphorylations of interest and results in significantly reduced signal. Switching to BSA may help to improve the strength of your band. Have you done any treatment as a positive control to ensure that this particular phosphorylation is expressed?

I hope this helps, if not, please let me know and I will be happy to help you further.